Most infectious agents cause disease via our mucosal surfaces, which also applies to biological warfare agents. Therefore, in developing a vaccine against infectious/biological warfare agents, it is important to induce responses that would act at mucosal surfaces, as well as in the systemic compartment. Francisella tularensis is a gram-negative pathogen and cause of tularemia. This microorganism is a "category A pathogen and biological warfare agent". The overall goal of this project is to develop a safe mucosal vaccine effective in inducing protective responses against infection by F. tularensis. Specifically, we will 1) Determine the immunogenicity of heat shock proteins of F. tularensis and the effect of the saponin analog GPI-0100 and of ricin B in modulating host immune responses following systemic or intranasal immunization of mice. Serum and secretions will be collected and assayed for the nature and level of antigen-specific antibody activity by ELISA. Cells will be cultured and assessed for antigen-specific T cell proliferative responses and cytokine production (by ELISA). The effectiveness of the response on protection will be assessed following systemic or mucosal challenge with F. tularensis. 2) Determine the cellular mechanism(s) by which F. tularensis and its LPS, and the adjuvants modulate host responses. The role of Toll-like receptors (TLRs) and the B7 costimulatory system in mediating host responses and infection will be assessed in vitro and in vivo. Antigen-presenting cells from normal and TLR deficient mice will be stimulated in vitro and assessed for changes in the expression of MHC and B7 by flow cytometry and for cytokine production by ELISA. The cell signaling pathways involved in cell activation will also be determined. TLR- and B7-knockout mice will be used to determine the role of TLRs and B7 in responses to F. tularensis and its LPS. Humoral and cellular responses will be assessed as indicated above. Clearance of F. tularensis will be measured by microbiologic analysis. 3) Derive and characterize genetically defined attenuated strains of F. tularensis LVS with mutations in the shikimate and/or purine metabolic pathways for use as a live vaccine. Mutants will be derived and tested in mice for their safety, persistence in host tissue by microbiologic analysis, immunogenicity by inducing cellular (cell proliferation and cytokine production) and humoral (nature and level of antibody activity by ELISA) responses, and effectiveness in inducing protective immunity. These studies will define the role of the innate and adaptive immune systems in inducing protective responses to F. tularensis and will define a safe and efficacious vaccine against mucosal or systemic challenge with F. tularensis.